CHARACTERISATION OF LIPOXYGENASE ACTIVITY
IN WALNUT CAKE
Olga Bińczak, Urszula Samotyja, Maria Małecka
Department of Food Commodity Science, Faculty of Commodity Science,
The University of Economics and Business, Poznan 61-875, Poland,
[email protected]
Abstract: Lipoxygenase (LOX) is an enzyme widely spread in plants and animals.
LOX is a family of iron-containing dioxygenases which catalyze the hydroperoxidation of lipids, containing a cis-cis-1,4-pentadiene structure. Although
this enzyme contributes to the creation of a pleasant aroma of certain plant materials,
its presence may also have a negative impact on food quality as it is supposed
to be a major cause of off-flavour development. Walnut cake, which is a by-product
of the cold pressing of oil process, contains high amount of linoleic and α-linolenic
acids what suggests that LOX activity in this material during storage is highly
possible. The aim of this study was to characterize the activity of LOX from walnut
cake, regarding pH optima and the enzyme dilution in the reaction mixture.
The research material was walnut cake, purchased from a local company. A series
of buffers in the pH range of 4.0-10.5 were prepared to evaluate the optimal pH
of LOX action. Enzyme activity was assessed in linoleic acid emulsion
by spectrophotometric measurements of the conjugated dienes formation
at λ=234 nm. The results show that the optimum pH occurs at 7.6, which indicates
that walnut LOX is of the II type. The effect of LOX extract dilution on its activity
was also studied.
Keywords: lipoxygenase, LOX, polyunsaturated fatty acids, optimum pH, walnuts,
lipid oxidation
INTRODUCTION
Lipoxygenase (LOX, linoleate; EC. 1.13.11.12) is an endogenous,
oxidizing enzyme which has been investigated in plants since 1932 [André
and Hou 1932]. Its presence has been detected in the cells of baker's yeast,
algae, fungi, cyanobacteria, mosses and more than 60 species and various
organs of plants [Brash 1999, Seth et al. 2009]. Moreover, in mammalian tissue
this enzyme is involved in biosynthesis of leukotrienes and lipoxins [Schewe
et al. 1986]. LOX belongs to the oxidoreductases class which enzymes catalyse
7
process of incorporation two oxygen atoms into the substrate molecule.
It is a metalloprotein, composed of a single polypeptide chain with 75-100·103
Da molecular weight, which contains non-heme bound iron ion (Fe2+)
in the active center [Baraniak and Szymanowska 2006]. The LOX could have
activity of dioxygenase, hydroperoxidase (secondary reaction conversion
of lipid hydroperoxides) and leukotriene synthase [Seth et al. 2009].
Under physiological conditions the LOX action primarily refers to catalysis
of the dioxidation of polyunsaturated fatty acids (PUFA) containing cis-cis1,4-pentadiene structure to produce conjugated diene hydroperoxides.
Although most of LOX`s require the presence of free fatty acids with
an activated methylene group between the two double bonds such as linoleic
and α-linolenic acid, other molecules containing none of the pentadiene moiety
(12-keto-(9Z)-octadecenoic acid) or methyl esters have been reported to serve
as substrates [Robinson et al. 1995]. Moreover, LOX from soybean
and cucumber root are exceptions because they are also act on bound fatty
acids [Matsui et al. 1998]. Oleic acid is not oxidized. The primary products
of reactions catalysed by LOX are 9-and 13-hydroperoxides, may be converted
both chemically and enzymically into number of secondary products. Shibata
et al. [1995], according to the structural similarity of proteins, classified plant
LOX`s genes into two types: I and II. For the first type the optimum operating
conditions are alkaline region (pH approximate pH 9.0), and the main products
of the reaction are 13- hydroperoxides. Type II is active at neutral pH,
and the products of actions are 13- and in addition 9-hydroperoxides.
They also possess the transit peptide responsible for protein transport
to chloroplasts.
Pumpkin, potatoes, tomatoes, beans, green pea, sweet lupine, cucumbers,
almonds and walnuts are only a few examples of plants in which presence of
LOX was founded. However, the best characterized enzyme is LOX from
soybean, even though it physiological roles are not completely known [Baysal
and Demirdöven 2007]. Four isozymes from this plant have been isolated:
LOX-1 belongs to type I, LOX-2, -3 and -4 belong to type II. LOX-1
with optimum at pH 9.0 requires the presence of free PUFA and forms 9and 13- hydroperoxides in the ratio of 1: 9. LOX-2 exhibits the highest activity
at pH 6.8, acting on triacylglycerols and free fatty acids, which forms 9and 13- hydroperoxides in the ratio 1:1 [Robinson et al. 1995]. In addition,
under anaerobic conditions, it can also cooxidize carotenoids and chlorophyll,
thus contributing to the bleaching reaction of raw materials.
8
Further isoenzymes are LOX-3, whose activity (as opposed to LOX-2)
is inhibited by calcium ions and LOX-4, which can be isolated by
electrophoresis or gel chromatography [Baysal and Demirdöven 2006].
LOX is activated by hydroperoxides (high spin state ion Fe2 + is oxidized
to Fe3+), and then the enzyme catalyses the processes, which consist of three
main steps: 1) the stereo-specific separation of hydrogen from a methylene
group, located between the double bonds and the consequent creation of a fatty
acid radical, 2) conversion of this radical to conjugated dienes, 3) the stereospecific insertion of oxygen into substrate molecules with formation
of the hydroperoxide [Baraniak and Szymanowska 2006]. These reactions
can be carried out under anaerobic or aerobic conditions, although they may
occur simultaneously. However, derivatives of the fatty acid hydroperoxides
are potentially responsible for aging of plant cells, deleterious to membrane
function by causing increased rigidity or formation epoxides. Furthermore,
primary products generated by the activity of LOX, act with other enzymes
such as lyase, isomerase and dehydrogenase and thus they are converted to
ketones, aldehydes and esters which has similar flavor volatiles to those
produced during autoxidation [Gardner 1991; Baysal and Demirdöven 2007].
At the same time hydroperoxides and hydroxy derivatives of unsaturated fatty
acids inhibit the action of certain enzymes and they have antibacterial
and antifungal functions [Grechkin 1998].
Due to wide presence in plant tissues, conducting the oxidation reaction,
the ability to form reactive oxygen species, and its reactions with other food
ingredients LOX has a significant effect on the stability, taste and color
of plant-based products [Baysal and Demirdöven 2007]. Its action contribute
to reduced level of vitamins C and E, lutein, carotenoids and chlorophylls
[Casey and Hughes, 2004]. On the other hand it is an enzyme needed
to maintain an adequate resistance to fungal, bacterial infections and insect
attacks in plants [Seth et al. 2009]. Contribution to the creation of fragrances
in raw materials, including bananas, tomatoes and melons, is also positive role
of this enzyme. Compounds responsible for this process are secondary
products of fatty acids’ oxidation such as short chain alcohols, aldehydes
and 6-carbon compounds called "green" odor. However, the final products
of oxidation lead to the formation of a grassy undesirable off-flavor in stored
legumes products [Casey et al. 1999]. Due to its characteristics, LOX is also
used in food technology, where it improves the rheological properties of wheat
dough [Frazier et al. 1973] and causes the bleaching of flour [Casey et al.
9
1999]. Moreover, the level of LOX activity may be a good indicator
to determine the optimal conditions for blanching of green beans and pea
[Barret and Theerakulkait 1995].
Although LOX is an enzyme which has been found in variety
of the plant-based raw materials, widely described are only enzymes present
in the seeds of leguminous plants, including soybeans, green pea and sweet
lupins. There are only a few study dealt with the characterization of LOX
from almond and walnut, which presence in these raw materials has been
confirmed by Zacheo et al. [2000], Buranasompob et al. [2007] or Salcedo
et al. [2010]. Both walnuts and walnut cake, formed as a by-product of the cold
pressing of oil process, are a rich source of nutrients, biologically active
compounds, such as proteins, polyphenols and polyunsaturated fatty acids
[Martinez et al. 2010]. The high content of lipid fraction, in which almost 73%
are the linoleic and α-linolenic acids [Salcedo et al. 2010], has the positive
effect on the nutritional value of walnut products. However, together
with the relevant conditions of water activity it may contribute to adverse
changes in this material during storage. One of the most important among
that is the fat oxidation process, may be promoted by the activity
of endogenous enzyme- LOX, for which linoleic and α-linolenic acids
are the mainly substrates. Hydroperoxides, formed as a result of LOX activity,
are precursors of volatile compounds, which can significantly influence
on the quality of stored nuts [Gordon et al. 2011, Samotyja and Małecka,
accepted paper].
The scientific evidence concerning walnut LOX is scarce. Taking into
consideration the high nutritional value of walnut cake and suitable conditions
for LOX activity, the aim of this study was to determine the optimal conditions
for LOX action in walnut cake.
MATERIALS AND METHODS
Chemicals
Linoleic acid (≥98%), Tween 20 and disodium phosphate (≥99%) were
purchased from Sigma-Aldrich, USA; acetic (≥99.5%), boric and phosphoric
(≥85%) acids and petroleum ether were bought from Chempur (Poland),
sodium hydroxide from Stanlab (Poland). All reagents were analytical grade.
10
Materials
The research material was ground walnut cake obtained from the local
company.
Characterization of material
Water activity (aw) was determined according to ISO 21807:2004
standard, with the use of Novasina ms1-aw apparatus (Novasina, Switzerland).
Determination of aw is based on conductivity measurement of hygroscopic
crystals in the sensor enBSK- 3/PP. The samples were placed in a disposable
bowl and inserted into the sensor. The time at which the measurement
was stabilised was established as six minutes. The results represent the mean
value obtained from four measurements.
Moisture: the moisture content of walnut cake was determined according
to AOAC Official Method 925.09. Samples were dried at 105°C until
the constant weight was achieved and then the residue was weighed.
The results represent the mean value obtained from four measurements.
The crude fat content was carried out with the use of Randall SER 148
apparatus (Velp, Italy). The dry walnut cake was weighed into an extraction
thimble. Extraction process was conducted for 60 min with petroleum ether
with the crucible immersed in boiling solvent and 60 min of reflux washing.
The dry residue in the extraction cup was weighed after evaporation
of the solvent. The results are given as the mean value obtained from three
different extractions processes.
Enzyme extraction
Enzyme isolation was performed according to the method described
by Zacheo [Zacheo et al. 2000]. 0,5 g of walnut cake was suspended in 10 ml
of suitable buffer and LOX was extracted on the shaker for 20 min at room
temperature. Then the homogenate was cooled in a refrigerator for 1 hour
and centrifuged for 10 min (4500g). After that, supernatant was filtered under
reduced pressure by using a Büchner's funnel. Obtained extract was used
to further analysis. The extraction process was performed in duplicate.
Preparation of linoleic acid emulsion
As the substrate solution the emulsion of linoleic acid was used.
It was prepared according to Salcedo [Salcedo et al. 2010] by mixing
11
on magnetic stirrer 20 µl of linoleic acid with 200 µl of Tween 20
and completed with phosphate buffer (pH 6.0) up to 5 ml of the final volume.
Preparation of buffers
To determine the optimal condition of LOX activity the series of buffer
in the range of 4.0-10.5 were prepared. Buffers in the range 4.0-5.0 and 8.5
-10.5 were obtained by using a universal Britton and Robinson buffer:
appropriate amount of 0.2 M NaOH was added to 100 ml of aqueous solution
of acids (0.04 M acetic acid + 0.04 M phosphoric acid + 0.04 M boric acid).
LOX activity at pH 5.5-8.0 was examined with using phosphate buffer,
prepared by combining appropriate proportions of aqueous solutions
of disodium phosphate (0.067 M) and monopotassium phosphate (0.067 M).
LOX activity of the crude extract
LOX activity of the crude extract was determined by a spectrophotometric analysis based on the measurement of the increase of absorbance
at λ=234 nm over a 1 min induced by conjugated diene formation from linoleic
acid. The reaction mixture was prepared by adding 1.96 ml of reaction buffer,
20 µl substrate solution and 20 μl of crude extract. The blank sample
was prepared without the solution of enzyme. All the assay was carried out
at the room temperature. One unit of LOX activity [U] was defined as
an increase of absorbance by 0.001 after 1 minute at λ=234 nm.
Characterization of LOX
The assessment of extraction conditions for LOX activity of the crude extract
The assessment of extraction conditions influence on LOX activity was
performed by using the buffers of pH: 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5,
10.0, 10.5 to extract the crude enzyme. The buffer with the same value
of pH was used to enzyme extraction and to preparation of the reaction
mixtures. LOX activity of the crude extract was measured under the standard
conditions. The results are given as the mean value of crude LOX activity
obtained from six different measurements.
The assessment of optimal pH of the reaction mixture
The optimum pH of reaction mixture was determined by measuring
crude LOX activity in the pH range between 4.0–10.5. The enzyme extraction
was carried out always by using phosphate buffer (pH 7.0). The different buffer
12
was used to prepare reaction mixtures containing 20 µl of substrate solution
and 20 µl of enzyme extract. Activity of LOX was measured under standard
conditions. The results are given as the mean value of enzyme activity obtained
from three different measurements.
Dilution of crude enzyme in reaction mixtures
To determine the correlation between crude extract concentration
the reaction mixture and its activity, the enzymatic extract in phosphate buffer
(pH 7.0) was diluted in the proportions: 1/5, 2/5, 3/5 and 4/5. The assay was
carried out under optimal conditions (pH 7.6). The results are given
as the mean value of LOX activity of crude extracts obtained from four
different measurements.
RESULTS AND DISCUSSION
Moisture, water activity and crude fat content
In order to characterize of walnut cake, moisture, total content of crude
fat and water activity were determined. The obtained values are shown
in Table 1. The water activity equal to 0.343 and humidity in the order
of 6.1% of the research material are acceptable and close to the values obtained
for the raw nuts [Biernat et al. 2014; Martinez et al. 2014]. According to
Rahman and Labuza [1999] optimum moisture for product having a water
activity in the range of 0.07-0.35 is 2-15%. Although the relatively low values
of aw and moisture are noted, these differentiators may affect to chemical
and biological reactions occurring in the material. Consequently they may have
an adverse impact on the length of storage lifetime of the product. It should
be noted that the minimum value of water activity needed for enzymatic action
of LOX is 0.4 so it is highly possible that enzymatic lipid oxidation occurs
in material during ageing. In raw nuts 60% of dry weight is fat fraction [United
States Department of Agriculture] and it is 7-fold higher than in the research
material, wherein 8.5% the crude fat content was obtained.
13
Table 1. Characteristics of walnut cake
Characteristics
Measured value
Water activity
Moisture [%]
Fat [% d.m.]
0.34±0.001
6.1±0.03
8.5±0.04
Source: Autors’ own work
The effect of extraction conditions on the LOX activity of the crude extract
The correlation between LOX activity of the crude extract isolated from
walnut cake and conditions of enzyme extraction was presented in Figure 1.
There was relatively stable crude extract activity in the pH range 5.5 to 8.0
and the systematic, almost proportional, increase with increasing alkalinity
of the extraction solvent (above pH 8). It should be noted, that the walnut cake
is a rich source of nutrients [Mártinez et al. 2014], which may precipitate
in the alkaline pH during extraction and have significant effect on final results.
Obtained darker, than from extracts prepared at lower pH, color of filtrate
could also confirm the high influence of alkaline extraction solvent
on the assay.
3000
Activity [U]
2500
2000
1500
1000
500
0
5.5
6
6.5
7
7.7
8
pH
8.5
9
9.5
10
10.5
Fig. 1. The influence of extraction conditions on the LOX activity of the crude
extracts. Source: Author’s own work
14
The literature data concerning the influence of the extraction conditions
on LOX activity from walnut and other raw materials is scarce. Extraction
of the enzyme at pH 7 was conducted by Kubicka, Jędrychowski [2001],
who isolated LOX from the pumpkin seeds. Salas et al. [1999], Salcedo et al.
2010 and Gökmen et al. [2005] have used distilled water to extracted enzyme
respectively from walnuts and almond, olive oil and pea. Yoshie-Stark
and Wäsche [2004] and Stephanie et al. [2014] isolated LOX from various
types of lupine at pH 6.8. All the authors do not explain the reasons for using
such pH. Taking into consideration the above literature information, significant
changes in the extract color and the influence of alkaline pH on the assay,
it was decided that enzyme extraction to further research will be carried
out at constant pH 7.0.
The influence of reaction mixture pH on the LOX activity of the crude
extract
The base to differentiate the LOX type is pH value of its optimal activity
[Shibata et al. 1995]. In this research the optimum for enzyme action at pH 7.6.
was observed. It indicate the presence of LOX type-II in walnut cake,
with the highest activity at neutral pH [Baysal and Demirdöven 2007].
In the literature there are different data about the optimum of walnut LOX.
Kosary et al. [2009] reported that optimum for this LOX is at pH 7.7, but they
investigated enzyme activity also at pH 4.5 and 8.5. Buranasompob et al.
[2007] stated that optimal conditions for enzyme action from walnut
and almonds are at pH 7.0 but the range of tested pH was limited to four values.
It should be also noted that LOX in plants exhibited optimum
for its action in the range of pH 5.0-9.0 and enzyme activity depends
on the plant species, store conditions and cultivation: lupins - pH 6.0-8.0
[Yoshie-Stark and Wäsch 2004], soybean LOX-1 – pH 9, LOX-2 – pH 6.5
[Sheu and Chen 1991], green pea – pH 5.5-6.0 [Gökmen, Bahceci,
and Acar 2002; Szymanowska et al. 2009].
15
500
Activity [U]]
400
300
200
100
4
5
5.5
6
6.3
6.5
6.6
6.8
7
7.5
7.6
7.7
7.8
8
9
9.5
10
10.5
0
pH
Fig. 2. The LOX activity dependence on the pH of the reaction mixture. Source:
Author’s own work
Activity [U]
Effect of extract dilution on LOX activity
The influence of extract dilution on LOX activity was presented
in Figure 3. The dependence was found to be linear. The literature on this
subject is scarce. There are only a few studies which have reported on material.
Szymanowska et al. [2009] found linear relationship between the LOX activity
from pea seeds and its dilution in the reaction mixture. This directly
proportional relation is typical only for LOX-2 [Szymanowska et al. 2009]
what proves that LOX present in the walnut cake is of type-II.
400
350
300
250
200
150
100
50
0
0
20
40
60
Enzyme dilution [%]
80
100
Fig. 3. Enzyme dilution profile for LOX. Source: Author’s own work
16
CONCLUSION
This study revealed conducive conditions to action of LOX in walnut cake
such as presence of polyunsaturated fatty acids and low water activity.
The optimum of LOX activity at pH 7.6 and the linear influence of enzyme dilution
on its activity revealed the presence of LOX type-II. Such a finding is very
important for quality of walnut cake as LOX-II may affect both fatty acids
bound in triacylgycerols as well as free PUFA, yielding formation
of hydroperoxides.
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19
STRESZCZENIE
Lipooksygenaza (LOX) jest enzymem należącym do rodziny oksydoreduktaz,
posiadającym w centrum aktywnym jon żelaza. LOX katalizuje przede wszystkim
reakcje utleniania kwasów tłuszczowych zawierających układ wiązań cis-cis-1,4pentadienowych, tworząc w konsekwencji skoniugowane dieny. Obecność tego
enzymu przyczynia się jednocześnie do powstawania przyjemnych aromatów
niektórych surowców roślinnych, jak i do pogorszenia ich jakości, poprzez
uczestnictwo w tworzeniu jełkich zapachów szczególnie w czasie ich przechowywania. Wytłok z orzecha włoskiego, jako produkt uboczny procesu tłoczenia
na zimno oleju, charakteryzuje się wysoką zawartością kwasów linolowego
i α-linolenowego, co sugeruje, że istnieje wysokie prawdopodobieństwo aktywności
LOX w tym surowcu w czasie jego przechowywania. Celem pracy była
charakterystyka warunków dla optymalnej aktywności LOX mając na względzie pH
oraz stopień rozcieńczenia enzymu w mieszaninie reakcyjnej. Materiał badawczy
stanowił wytłok z orzecha włoskiego, otrzymany od lokalnego przedsiębiorstwa.
Przygotowano szereg buforów w zakresie pH 4.0-10.5. Aktywność enzymu mierzono
w emulsji kwasu linolowego, poprzez spektrofotometryczny pomiar wzrostu
absorbancji, przy długości fali równej 234 nm. Stwierdzono optimum dla aktywności
LOX przy pH równym 7.6, co wskazuje, że w surowcu występuje LOX typu-II.
Wykreślono krzywą zależności aktywności enzymu od jego stężenia w układzie
reakcyjnym.
Słowa kluczowe: lipooksygenaza, LOX, wielonienasycone kwasy tłuszczowe,
optimum pH, orzechy włoskie, utlenianie lipidów
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